i'm not sure i agree with you, deanne.
i don't think there is any means for the pre-selective amp to "specifically
amplify" Eco/Mse fragments. there is no control in either PCR to prevent
amplification of Mse/Mse fragments or Eco/Eco fragments (i've tried throwing
only Eco primers in the selective amp - i thought something was wrong with my
Mse primers so wanted a control - and saw tons of product on an agarose gel!).
so, i think toby might be onto something.
perhaps you want to try amplifying what you cut out of your silver stained gel
in an asymetrical PCR (normal or higher [Eco-NNN] and very low [Mse-NNN]). or
maybe you could skip the Mse primer altogether. that will give you single
stranded product but should certainly tell you it's an Mse-Mse fragment causing
you problems.
good luck!
tim
Deanne Bell wrote:
> Hi Toby
>> This is a nice thought, but the AFLP process goes through 2 amplifications:
> a) the pre-selective amplification sorts out all of the Eco/Eco & Mse/Mse
> fragments because it specifically amplifies the Eco/Mse fragments
> b) the second amplification (selective amp) "selects" sub-sets of those
> Eco/Mse fragments by extending the primers by 1 or 2 bases.
>> By the time the product is run out on a gel, I don't think any residual
> Eco/Eco or Mse/Mse fragments would be detectable.
> D.Bell
>> ----------
> : From: 'Toby' H D Bradshaw <toby at u.washington.edu>
> : To: gen-link at hgmp.mrc.ac.uk.methods@hgmp.mrc.ac.uk
> : Subject: Re: Unusual bands on silver stained AFLP
> : Date: Monday, May 14, 2001 11:51 AM
> :
> : Consider the possibility that your silver stain recognizes all AFLP bands
> : (Eco/Eco, Eco/Mse, Mse/Mse), while your radioactive bands are labeled on
> : the Eco primer only [if you are running conventional AFLPs as described
> by
> : Vos et al.]. The 'monomorphic' band may be an Mse/Mse fragment that you
> : have never seen with radioactivity, sitting on top of the polymorphic
> : Eco/Mse (usually) band.
> :
> : Of course I have no idea what sort of AFLP protocol you are using so this
>> : advice may be off base.
> :
> : Toby Bradshaw | (206)616-1796 (voice)
> : College of Forest Resources | (206)685-2692 (FAX)
> : University of Washington | http://poplar2.cfr.washington.edu/toby> :
> :
> : In article <3AFFED74.4471ED9C at ncsu.edu>, Liz Parks <liz_parks at ncsu.edu>
> wrote:
> : >Hello all,
> : >I am baffled by the banding pattern on my silver stained AFLP gel.
> : >Normally I run radioactive AFLP, and when I find some interesting bands,
> : >I run a non-radioactive reaction and silver stain it so that I can cut
> : >out the bands and reamplify them for sequencing or cloning. I've
> : >sequenced 100 bands this way, and it works really well. However, there
> : >is a polymorphic band that I am interested in, that has been confirmed
> : >in two separate radioactive AFLP reactions, that appears to be
> : >monomorphic on the silver stained gel. I repeated the PCR, and ran it a
> : >second time, with the same result. There are other polymorphic bands
> : >that appear on the gel, so I don't think contamination is the answer.
> : >There is a monomorphic band below the band of interest, and the bands
> : >are well resolved, but I wonder if one of the denatured strands migrates
> : >slower with the larger band, and this was not detected with radioactive
> : >AFLP. Any thoughts on what's going on, and how I can isolate my band?
> : >Thanks,
> : >Liz
> : >
> : >--
> : >
> : >~~~~~~~~~~~~~~~~~~~~~
> : >Liz Parks
> : >North Carolina State University
> : >Department of Plant Pathology
> : >Raleigh, NC 27695
> : >~~~~~~~~~~~~~~~~~~~~~
> : >
> : >
> :
> :
>> ---